Association of CYP7B1 expression with the prognosis of endometrial cancer: a retrospective study.

BACKGROUND: Endometrial cancer (EC) tissues express CYP7B1, but its association with prognosis needs to be investigated. METHODS: Immunohistochemistry and image analysis software were used to assess CYP7B1 protein expression in paraffin-embedded endometrial tumor sections. Associations between CYP7B1 and clinical factors were tested with the Wilcoxon rank-sum test. Kaplan-Meier curves were employed to describe survival, and differences were assessed using the log-rank test. Cox regression analysis was used to assess the association between CYP7B1 expression and the prognosis of patients with EC. RESULTS: A total of 307 patients were enrolled with an average age of 52.6 ± 8.0 years at diagnosis. During the period of follow-up, 46 patients (15.0%) died, and 29 (9.4%) suffered recurrence. The expression of CYP7B1 protein is significantly higher in the cytoplasm than in the nucleus (P < 0.001). Patients aged < 55 years (P = 0.040), ER-positive patients (P = 0.028) and PR-positive patients (P < 0.001) report higher levels of CYP7B1 protein. Both univariate (HR = 0.41, 95% CI: 0.18-0.90, P = 0.025) and multivariate (HR = 0.35, 95%CI:0.16-0.79, P = 0.011) Cox regression analyses demonstrate that high CYP7B1 protein expression predicts longer overall survival (OS). When considering only ER-positive patients (n = 265), CYP7B1 protein expression is more strongly associated with OS (HR = 0.20,95%CI:0.08-0.52, P = 0.001). The 3-year OS and 5-year OS in the low-CYP7B1 subgroup are 81.6% and 76.8%, respectively; while in the high-CYP7B1 subgroup are 93.0% and 92.0%, respectively (P = 0.021). CONCLUSIONS: High CYP7B1 protein expression predicted longer OS, suggesting that it may serve as an important molecular marker for EC prognosis.

Machine learning-based analysis identifies and validates serum exosomal proteomic signatures for the diagnosis of colorectal cancer.

The potential of serum extracellular vesicles (EVs) as non-invasive biomarkers for diagnosing colorectal cancer (CRC) remains elusive. We employed an in-depth 4D-DIA proteomics and machine learning (ML) pipeline to identify key proteins, PF4 and AACT, for CRC diagnosis in serum EV samples from a discovery cohort of 37 cases. PF4 and AACT outperform traditional biomarkers, CEA and CA19-9, detected by ELISA in 912 individuals. Furthermore, we developed an EV-related random forest (RF) model with the highest diagnostic efficiency, achieving AUC values of 0.960 and 0.963 in the train and test sets, respectively. Notably, this model demonstrated reliable diagnostic performance for early-stage CRC and distinguishing CRC from benign colorectal diseases. Additionally, multi-omics approaches were employed to predict the functions and potential sources of serum EV-derived proteins. Collectively, our study identified the crucial proteomic signatures in serum EVs and established a promising EV-related RF model for CRC diagnosis in the clinic.

LILRB4 Checkpoint for Immunotherapy: Structure, Mechanism and Disease Targets.

LILRB4, a myeloid inhibitory receptor belonging to the family of leukocyte immunoglobulin-like receptors (LILRs/LIRs), plays a pivotal role in the regulation of immune tolerance. LILRB4 primarily mediates suppressive immune responses by transmitting inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs (ITIMs). This immune checkpoint molecule has gained considerable attention due to its potent regulatory functions. Its ability to induce effector T cell dysfunction and promote T suppressor cell differentiation has been demonstrated, indicating the therapeutic potential of LILRB4 for modulating excessive immune responses, particularly in autoimmune diseases or the induction of transplant tolerance. Additionally, through intervening with LILRB4 molecules, immune system responsiveness can be adjusted, representing significant value in areas such as cancer treatment. Thus, LILRB4 has emerged as a key player in addressing autoimmune diseases, transplant tolerance induction, and other medical issues. In this review, we provide a comprehensive overview of LILRB4, encompassing its structure, expression, and ligand molecules as well as its role as a tolerance receptor. By exploring the involvement of LILRB4 in various diseases, its significance in disease progression is emphasized. Furthermore, we propose that the manipulation of LILRB4 represents a promising immunotherapeutic strategy and highlight its potential in disease prevention, treatment and diagnosis.

M1 polarization of macrophages promotes stress-induced hair loss via interleukin-18 and interleukin-1ß.

Stress-induced hair loss is a prevalent health concern, with mechanisms that remain unclear, and effective treatment options are not yet available. In this study, we investigated whether stress-induced hair loss was related to an imbalanced immune microenvironment. Screening the skin-infiltrated immune cells in a stressed mouse model, we discovered a significant increase in macrophages upon stress induction. Clearance of macrophages rescues mice from stress-induced hair shedding and depletion of hair follicle stem cells (HFSCs) in the skin, demonstrating the role of macrophages in triggering hair loss in response to stress. Further flow cytometry analysis revealed a significant increase in M1 phenotype macrophages in mice under stressed conditions. In searching for humoral factors mediating stress-induced macrophage polarization, we found that the hormone Norepinephrine (NE) was elevated in the blood of stressed mice. In addition, in-vivo and in-vitro studies confirm that NE can induce macrophage polarization toward M1 through the ß-adrenergic receptor, Adrb2. Transcriptome, enzyme-linked immunosorbent assay (ELISA), and western blot analyses reveal that the NLRP3/caspase-1 inflammasome signaling and its downstream effector interleukin 18 (IL-18) and interleukin 1 beta (IL-1ß) were significantly upregulated in the NE-treated macrophages. However, inhibition of the NE receptor Adrb2 with ICI118551 reversed the upregulation of NLRP3/caspase-1, IL-18, and IL-1ß. Indeed, IL-18 and IL-1ß treatments lead to apoptosis of HFSCs. More importantly, blocking IL-18 and IL-1ß signals reversed HFSCs depletion in skin organoid models and attenuated stress-induced hair shedding in mice. Taken together, this study demonstrates the role of the neural (stress)-endocrine (NE)-immune (M1 macrophages) axis in stress-induced hair shedding and suggestes that IL-18 or IL-1ß may be promising therapeutic targets.

Novel role of immune-related non-coding RNAs as potential biomarkers regulating tumour immunoresponse via MICA/NKG2D pathway.

Major histocompatibility complex class I related chain A (MICA) is an important and stress-induced ligand of the natural killer group 2 member D receptor (NKG2D) that is expressed in various tumour cells. Given that the MICA/NKG2D signalling system is critically embedded in the innate and adaptive immune responses, it is particularly involved in the surveillance of cancer and viral infections. Emerging evidence has revealed the important roles of non-coding RNAs (ncRNAs) including microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in different cancer types. We searched for all relevant publications in the PubMed, Scopus and Web of Science database using the keywords ncRNA, MICA, NKG2D, cancer, and miRNAs. All relevant studies published from 2008 to the 2023 were retrieved and collated. Notably, we found that miRNAs can target to NKG2D mRNA and MICA mRNA 3'-untranslated regions (3'-UTR), leading to translation inhibition of NKG2D and MICA degradation. Several immune-related MICA/NKG2D pathways may be dysregulated in cancer with aberrant miRNA expressions. At the same time, the competitive endogenous RNA (ceRNA) hypothesis holds that circRNAs, lncRNAs, and mRNAs induce an abnormal MICA expression by directly targeting downstream miRNAs to mediate mRNA suppression in cancer. This review summarizes the novel mechanism of immune escape in the ncRNA-related MICA/NKG2D pathway mediated by NK cells and cancer cells. Moreover, we identified the miRNA-NKG2D, miRNA-MICA and circRNA/lncRNA/mRNA-miRNA-mRNA/MICA axis. Thus, we were particularly concerned with the regulation of mediated immune escape in the MICA/NKG2D pathway by ncRNAs as potential therapeutic targets and diagnostic biomarkers of immunity and cancer.

Clinicopathological features and immunophenotype of Silva pattern system in endocervical adenocarcinoma.

The aim of this study was to investigate the correlation between Silva pattern system and clinicopathological features of endocervical adenocarcinoma. Moreover, it was to find molecular markers helpful for Silva classification, and thus we also explored the expression levels of invasion, adhesion and proliferation biomarkers in cases of Silva non-invasive and invasive types. The survival based on Silva pattern system was analysed by Kaplan-Meier survival analysis, Log-rank test and  a COX risk proportionality model. Sixty samples were chosen to detect the MMP-2, MMP-9, u-PA, E-cadherin, ß-catenin, EGF, TGF-α, HDGF, c-Met and RGN expression by immunohistochemistry. Multivariate analysis showed that pattern A/pattern B/pattern C Silva pattern system provided independent risk factors for prognosis. Our results found the levels of MMP-2, MMP-9 and u-PA were significantly higher in endocervical adenocarcinoma with destructive growth than in the  nondestructive group. The levels of E-cadherin and ß-catenin were significantly lower in endocervical adenocarcinoma with destructive growth than in the nondestructive group. The levels of EGF, TGF-α and HDGF were significantly higher in endocervical adenocarcinoma with destructive growth than in the nondestructive group. Compared with 'non-invasive/invasive Silva pattern', this study suggests 'pattern A/pattern B/pattern C Silva pattern' could be a better criteria for predicting the prognosis. Furthermore, the dual-marker combination of 'MMP-2 and u-PA' and 'E-cadherin and ß-catenin' is very important in the diagnosis of Silva pattern classification.

IGF2BP2 Promotes Epithelial to Mesenchymal Transition and Metastasis through Stabilizing HMGA1 mRNA in Gastric Cancer.

As an RNA-binding protein, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is involved in enhancing the progression of a few malignant tumors by recognizing N6-methyladenosine on targeted RNA. However, the specific effects of IGF2BP2 on gastric cancer (GC) and the underlying mechanisms remain unclear. In this study, the expression level of IGF2BP2 was evaluated by analyzing data from a public database and performing immunohistochemical staining with GC specimens. The effect of IGF2BP2 on GC cell metastasis was investigated by Transwell assays and animal studies. RNA immunoprecipitation (RIP) was performed to identify potential mRNA bound to IGF2BP2. The levels of these identified RNAs were measured by RT-PCR, while corresponding proteins were quantified via Western blot. It was revealed that IGF2BP2 expression in GC tissues was significantly upregulated, and its overexpression was significantly associated with worse survival in GC patients. The aberrant expression of IGF2BP2 was demonstrated to promote the invasion and metastasis of GC cells by both in vivo and in vitro experiments. In subsequent experiments, it was then verified that by directly interacting with HMGA1 mRNA, IGF2BP2 augmented its stability and thus increased its expression. The knocking down of IGF2BP2 could significantly decrease the migration and invasion of GC cells, which could be reversed by increasing HMGA1 expression. Additionally, both in vitro and in vivo epithelial-mesenchymal transition (EMT) of GC cells were enhanced by IGF2BP2/HMGA1 axis. In conclusion, it was proven in our study that the IGF2BP2/HMGA1/EMT axis contributed to GC metastasis, suggesting its potential as a novel predictive and therapeutic biomarker for GC.

A Soluble NK-CAR Mediates the Specific Cytotoxicity of NK Cells toward the Target CD20+ Lymphoma Cells.

The structures of chimeric antigen receptors (CARs) currently designed for natural killer (NK) cells are mostly based on knowledge gained about CAR-T cells. Although these CAR-NK cells have shown promising effects, there are still many limitations to their application. In this study, we designed a soluble NK-CAR since the membrane protein NKG2D expressed by NK cells can directly trigger NK cell cytotoxicity by binding with the ligand MICA. This CAR is composed of three segments: the extracellular domain of MICA, an anti-CD20 single-chain variable fragment (anti-CD20 ScFv), and a human IgG Fc component. The nucleotide sequence of the soluble NK-CAR was cloned into a eukaryotic expression vector and expressed in suspension HEK293 cells, and the recombinant NK-CAR protein was then purified in a Staphylococcus aureus protein A column. The novel NK-CAR exhibited bifunctional activity, recognizing both the CD20 antigen of target cells and the NKG2D receptor of NKL cells. The NK-CAR activated the NKG2D receptor signaling pathway, causing NKL cells to express CD107a and secrete interferon-gamma. The soluble NK-CAR mediated the NKL cell killing of CD20+ Daudi cells in vitro, with a 1 µg/mL concentration inducing the maximum killing effect. Moreover, 51.7% (p < 0.01) of Daudi cells were killed at the effector-to-target ratio of 10:1. In the presence of recombinant rMICA and NKG2D-Ig proteins, this killing effect was reduced to 30% (P < 0.01) owing to competitive interference. Our results highlight the clinical application potential of this novel immunotherapy for killing target tumor cells.

Organoids From Mucinous Appendiceal Adenocarcinomas as High-Fidelity Models for Individual Therapy.

Background: Mucinous appendiceal adenocarcinoma (MAA) is a rare, heterogeneous disease. Patients with unrespectable mucinous appendiceal adenocarcinoma presenting with peritoneal spread are treated by intraperitoneal chemotherapy, hyperthermic intraperitoneal chemotherapy, systemic chemotherapy, or targeted therapy. However, there are no guidelines for efficacious drugs against mucinous appendiceal adenocarcinoma. Therefore, relevant high-fidelity models should be investigated to identify effective drugs for individual therapy. Methods: Surgical tumor specimens were obtained from a mucinous appendiceal adenocarcinoma patient. The tissue was digested and organoid culture was established. H&E and immunohistochemistry staining as well as DNA sequencing was performed on tissue and organoid. The pathological characteristics and gene mutations of the organoid were compared to those of the original tumor. Drug sensitivity tests were performed on organoid and the patient clinical responds to chemotherapy and targeted therapy was compared. Results: Organoids were successfully established and stably passaged. Pathological characteristics of organoids including H&E staining and expression of protein markers (CK20, CDX-2, STAB2, CD7, PAX8) were consistent to those of the original tumor. Moreover, the organoids carried the same gene mutations as the primary tumor. Sensitivity of the organoids to chemotherapeutic drugs and tyrosine kinase inhibitors included: 5-FU (IC50 43.95 µM), Oxaliplatin (IC50 23.49 µM), SN38 (IC50 1.02 µM), Apatinib (IC50 0.10 µM), Dasatinib (IC50 2.27 µM), Docetaxel (IC50 5.26 µM), Regorafenib (IC50 18.90 µM), and Everolimus (IC50 9.20 µM). The sensitivities of organoid to these drugs were comparable to those of the patient's clinical responses. Conclusion: The mucinous appendiceal adenocarcinoma organoid model which retained the characteristics of the primary tumor was successfully established. Combined organoid-based drug screening and high throughput sequencing provided a promising way for mucinous appendiceal adenocarcinoma treatment.

Tereticornate A suppresses RANKL-induced osteoclastogenesis via the downregulation of c-Src and TRAF6 and the inhibition of RANK signaling pathways.

Excessive osteoclast differentiation and activation are closely associated with the development and progression of osteoporosis. Natural plant-derived compounds that can inhibit osteoclastogenesis are an efficient strategy for the prevention and treatment of osteoporosis. Tereticornate A (TA) is a natural terpene ester compound extracted from the leaves and branches of Eucalyptus gracilis, with antiviral, antibacterial, and anti-inflammatory activities. However, the effect of TA on osteoclastogenesis and the underlying molecular mechanism remain unclear. Based on the key role of the NF-κB pathway in the regulation of osteoclastogenesis and the observation that TA exhibits an anti-inflammatory effect by inhibiting NF-κB activity, we speculated that TA could exert anti-osteoclastogenesis activity. Herein, TA could inhibit the RANKL-induced osteoclast differentiation and formation of F-actin rings in RAW 264.7 cells. Mechanistically, TA downregulated the expression of c-Src and TRAF6, and also suppressed the RANKL-stimulated canonical RANK signaling pathways, including AKT, MAPK (p38, JNK, and ERK), and NF-κB; ultimately, downregulating the expression of NFATc1 and c-Fos, the key transcriptional factors required for the expression of genes (e.g., TRAP, cathepsin K, ß-Integrin, MMP-9, ATP6V0D2, and DC-STAMP) that govern osteoclastogenesis. Our findings demonstrated that TA could effectively inhibit RANKL-induced osteoclastogenesis via the downregulation of c-Src and TRAF6 and the inhibition of RANK signaling pathways. Thus, TA could serve as a novel osteoclastogenesis inhibitor and might have beneficial effects on bone health.

Detection of the 23-bp nucleotide sequence mutation in retinoid acid receptor related orphan receptor alpha (RORA) gene and its effect on sheep litter size.

Retinoid acid receptor related orphan receptor alpha (RORA) transcribes steroid-related genes to regulate estrogen synthesis. As an important reproductive trait, litter size relates to estrogen synthesis. Therefore, it is important to investigate the association between RORA gene and sheep litter size. In this study, one 23-bp nucleotide sequence mutation was identified in intron 1 of RORA gene in 532 female Australian White Sheep. Moreover, the polymorphic information content (PIC) values of this locus was 0.219. The litter size of ID genotype was significantly better than II genotype and DD genotype in the second born litter size (p < 0.05). This loci was related to third born litter size and the ID is the dominant genotype (p < 0.05). The association between combined genotypes and average litter size showed that sheep with heterozygote (ID) genotypes had larger lamb than homozygous (DD and II) genotypes. To sum, this study provided theoretical references for the comprehensively research of the function of RORA gene and the breeding of Australian White Sheep. The 23-bp indel variants could be considered as molecular markers for the second and third born litter size of sheep for MAS breeding.

YAP1 induces marrow derived suppressor cell recruitment in Chlamydia trachomatis infection.

Chlamydia trachomatis (C. trachomatis) is the most commonly diagnosed bacterial sexually transmitted infection (STI) worldwide. Marrow derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes, which are effective inhibitors for T cell activation. This study explores the role of MDSCs in the immune escape mechanism of C. trachomatis. We established a vaginal infection model of a BALB/c-Chlamydia trachomatis mouse pneumonia strain (MoPn), and compared the percentages of MDSCs, CD4+T, and CD8+T cells in the spleen and cervix of mice before and after infection. The expression levels of arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) in MDSCs, and the expression level of transcriptional co-activator yes-associated protein 1 (YAP1) in the cervix were also compared. The results show that the proportion of MDSCs increases, while the proportion of CD4+T and CD8+T cells decreases after C. trachomatis-infection. The expression of Arg-1 and iNOS in MDSCs and YAP1 in host cells is up-regulated. C. trachomatis growth is inhibited after the inhibition of YAP1 in host cells. The proportion of MDSCs decreases after in vivo pharmacological inhibition of YAP1 in the C. trachomatis-infected mouse model. These results demonstrate, for the first time, the participation of MDSC in the immune escape of C. trachomatis under the action of YAP1.

Down-regulation of SOCS6: an unfavorable prognostic factor for gastrointestinal stromal tumor proven by survival analysis.

BACKGROUND: Many studies reporting that down-regulation of SOCS6 plays vital roles in promoting progression of malignant tumors have been published. The present study was performed to evaluate whether SOCS6 was significantly associated with prognosis of GIST patients. METHODS: Immunohistochemical staining was accomplished to evaluate the expression levels of SOCS6 among GIST patients. The impacts of SOCS6 expression on overall survival (OS) and recurrence-free survival (RFS) of GIST patients were assessed by Cox proportional hazard regression model analysis and Kaplan-Meier curve analysis. RESULTS: It was demonstrated that the expression level of SOCS6 was significantly associated with tumor size (P=0.001). Then according to Kaplan-Meier curve analysis, low expression of SOCS6 was significantly correlated with worse OS and RFS of GIST patients. Ultimately, it was revealed by Cox proportional regression model analysis that low expression of SOCS6 was an independent predictive factor for OS and RFS. CONCLUSIONS: Low expression of SOCS6 was an independent prognostic factor for GIST, suggesting its potential as a novel biomarker predicting survival of GIST patients.

Tumor-associated macrophages induced spheroid formation by CCL18-ZEB1-M-CSF feedback loop to promote transcoelomic metastasis of ovarian cancer.

BACKGROUND: Ovarian cancer (OvCa)-tumor-associated macrophages (TAMs) spheroids are abundantly present within ascites of high malignant patients. This study investigated the mutual interaction of OvCa cells and TAMs in the spheroids. METHODS: Three-dimensional coculture system and transwell coculture system were created to mimic the OvCa and TAMs in spheroids and in disassociated state. Transwell-migration assay and scratch wound healing assay were used to measure the invasive and migratory capacity. Western blot, quantitative reverse transcription-PCR and immunostaining were used to measure the mesenchymal and epithelial markers. Flow cytometry was used to assess the polarization of TAMs. Also, the differential gene expression profile of OvCa cells and OvCa cells from spheroids were tested by RNA-sequence. Finally, the ovarian mice models were constructed by intraperitoneal injection of ID8 or OvCa-TAMs spheroids. RESULTS: Our results indicated that the formation of OvCa-TAMs spheroids was positive related to the malignancy of OvCa cells. M2-TAMs induced the epithelial-mesenchymal transition of OvCa cells by releasing chemokine (C-C motif) ligand 18 (CCL18) in the spheroids. While, CCL18 induced macrophage colony-stimulating factor (M-CSF) transcription in OvCa cells through zinc finger E-box-binding homeobox 1 (ZEB1). This study further indicated that M-CSF secreted by OvCa cells drived the polarization of M2-TAMs. Therefore, a CCL18-ZEB1-M-CSF interacting loop between OvCa cells and TAMs in the spheroids was identified. Moreover, with blocking the expression of ZEB1 in the OvCa cell, the formation of OvCa-TAMs spheroids was impeded. In the ovarian mice models, the formation of OvCa-TAMs spheroids in the ascites was promoted by overexpressing of ZEB1 in OvCa cells, which resulted in faster and earlier transcoelomic metastasis. CONCLUSION: These findings suggested that the formation of OvCa-TAMs spheroids resulted in aggressive phenotype of OvCa cells, as a specific feedback loop CCL18-ZEB1-M-CSF in it. Inhibition of ZEB1 reduced OvCa-TAMs spheroids in the ascites, impeding the transcoelomic metastasis and improving the outcome of ovarian patients.

An efficient and user-friendly method for cytohistological analysis of organoids.

Organoid culture is a recently developed in vitro three-dimensional (3D) cell culture technology. It has wide applications in tissue engineering studies. However, histological analysis of organoid is quite complex and tedious for researchers. This study proposes a user-friendly, affordable and efficient method for making formalin-fixed paraffin embedded (FFPE) organoid blocks and Optimal Cutting Temperature compound (OCT) embedded frozen organoid blocks. This method implements a key pre-embedding step for preparing paraffin embedded organoid blocks, which could concentrate organoid together without damaging or loss of samples. This method could be used to process even a small number of organoids with high efficiency. In addition, with minor modifications, the method is readily applied for OCT embedded organoid blocks. The slides generated were ready for H&E staining, immunohistochemistry staining and immunofluorescent staining. The method described in this study can be easily used for routine histological analysis of organoid, and could be performed in general pathology labs and requires no dedicated equipment and reagent.

Polysaccharides from Chrysanthemun indicum L. enhance the accumulation of polysaccharide and atractylenolide in Atractylodes macrocephala Koidz.

Atractylodes macrocephala Koidz. (AM), an herb of traditional Chinese medicine, is well-known for anti-oxidant, anti-tumor and immune regulation potential. However, it is low bioactive compound content that restricts the application of this species. Elicitation is considered as an effective method to enhance biomass and bioactive compound in plants. Our precious study found that polysaccharide of Chrysanthemun indicum L. could promote plant growth by triggering plant defense. In the present study, polysaccharide of Chrysanthemun indicum L. is used to stimulate the accumulation of biomass and bioactive compound with different concentration in Atractylodes macrocephala Koidz. during pot, plot and field experiments. The results suggested that polysaccharide of Chrysanthemun indicum L. could significantly enhance the accumulation of biomass, atractylenolides and polysacchrides. Moreover, 2 mg/mL is determined and verified to be the appropriate concentration during field experiments. In addition, RT-qPCR revealed that CIP-induced terpenoid synthesis in AM mainly depended on mevalonate (MVA) pathway. This is the first report on the discovery of polysaccharide of Chrysanthemun indicum L. for the enhanced accumulation of biaomass and bioactive compound and the use of its for agricultural production.

The prognostic impacts of PABPC1 expression on gastric cancer patients.

Aim: To assess the prognostic impacts of PABPC1 on gastric cancer (GC) patients. Methods: The expression levels of PABPC1 in GC tissues and normal gastric tissues were initially compared via bioinformatics analysis. Immunohistochemical staining was accomplished to assess the expression of PABPC1 in the included GC patients. Then the impacts of PABPC1 expression on survival of GC patients were evaluated by Cox regression and Kaplan-Meier analyses. Results: The expression levels of PABPC1 in gastric tissues were significantly higher than those in normal gastric tissues (paired, p = 0.002; unpaired, p = 3.60e-9). By Kaplan-Meier, it was demonstrated that high expression of PABPC1 was significantly associated with worse overall and disease-free survival. Furthermore, high PABPC1 expression was demonstrated to be an independent predictive factor for both overall (p = 0.013; hazard ratio = 2.058; 95% CI: 1.162-3.644) and disease-free (p = 0.018; hazard ratio = 2.284; 95% CI: 1.153-4.524) survival. Conclusion: PABPC1 is a potential prognostic biomarker for GC patients.

Lay abstract Previous studies have reported that PABPC1 is involved in a series of biological processes and participates in many cancers. However, the specific role of PABPC1 in different cancers varies significantly, and PABPC1 has not been fully investigated in gastric cancer (GC). In the present study, it was demonstrated that PABPC1 was significantly upregulated in GC and its high expression in GC was significantly associated with worse overall and disease-free survival, indicating the potential of PABPC1 as a novel prognostic biomarker for GC.

[Separation and identification of impurities from intermediates of istradefylline].

Istradefylline is a novel selective adenosine A2A receptor antagonist that is used to treat Parkinson's disease and improve motor dysfunction in the early stage of this disease. During the synthesis of intermediate A1 (6-amino-1,3-diethyl-2,4-(1H,3H)-pyrimidinedione), at least two by-products were formed under alkaline or high-temperature conditions. In a previous study, one of the by-products in the synthesis of the intermediate was studied, and its structure was identified as (E)-N-ethyl-2-cyano-3-ethylamino-2-butene amide. In this study, we used high performance liquid chromatography (HPLC) to analyze another impurity formed during the synthesis of A1, and the following steps were executed: 0.4 g of intermediate was weighed and added to a 50 mL beaker, followed by the sequential addition of 8 mL water and 8 mL acetonitrile, and then, ultrasonic dissolution was performed. Finally, the solution was filtered through a 0.45-µm organic membrane and the test sample solution was obtained. We used the Agilent zorbax C18 chromatography column, with acetonitrile (A)/water(B) as the mobile phase under gradient elution ((tmin/A∶B)=t0/20∶80, t15/60∶40, t20-t50/90∶10). The detector wavelength is 268 nm. In order to separate the impurity from A1, we used a Ceres B preparative column, with acetonitrile-water (30/70, v/v) as the mobile phase. The flow rate was set at 30 mL/min, and the detection wavelength was 268 nm. The structure of the impurity was confirmed by high-resolution mass spectrometry (HRMS), one-dimensional nuclear magnetic resonance (NMR), and two-dimensional nuclear magnetic resonance (2D NMR), and characterized by single-crystal X-ray diffraction (XRD). In MS experiments, an electrospray ionization (ESI) source was used with positive ion scanning. In the NMR experiments, we used tetramethylsilane (TMS) as the internal standard and deuterated dimethyl sulfoxide (DMSO-d6) as the solvent to obtain the spectra. The results of preparative high performance liquid chromatography (Prep-HPLC) showed that good separation effect could be achieved by isocratic elution, and the impurity was perfectly separated. The1H-NMR spectral data are as follows:1H-NMR (600 MHz, DMSO): δ 1.01 (q, J=6.9 Hz, 3H), 1.02 (q, J=6.9 Hz, 3H), 1.07 (t, J=6.9 Hz, 3H), 3.04 (p, J=6.8 Hz, 2H), 3.74 (q, J=7.0 Hz, 2H), 3.94 (q, J=7.1 Hz, 2H), 5.85 (s, 1H). The 13C-NMR spectral data are summarized as follows: 13C-NMR (150 MHz, DMSO): δ13.9, 14.1, 15.9, 34.6, 34.9, 36.9, 81.9, 152.2, 153.3, 159.3, 162.0. The impurity was characterized by single-crystal XRD, and its spatial structure was further verified and determined as 1-(1,3-diethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-3-ethylurea. Based on the chemical structure of the impurity, we propose the following mechanism for the impurity: when A1 is synthesized under alkaline conditions or at high temperature, excessive diethylurea continues to undergo amidation with A1 to obtain this by-product. Although the formation mechanism of the impurity studied in this paper is different from that of the intermediate A1 impurity (E)-N-ethyl-2-cyano-3-ethylamino-2-butene amide, both the impurities are formed at high temperatures. Both will be accompanied by A1 in the subsequent reaction of istradefylline synthesis. The relationship between drug impurities and drug safety is a complex relationship that is affected by many factors. Generally, most impurities in drugs have potential biological activities, and some even interact with the drugs, thus affecting their efficacy and safety and inducing toxic effects. Therefore, to ensure the quality of istradefylline, it is necessary to control the impurity content during the production. The findings of this paper may provide guidelines for controlling the impurity content in istradefylline.

Laparoscopic gastrectomy plus D2 lymphadenectomy is as effective as open surgery in terms of long-term survival: a single-institution study on gastric cancer.

BACKGROUND: Laparoscopic surgery has been widely accepted to treat early-stage gastric cancer. However, it is still controversial to perform laparoscopic gastrectomy plus D2 lymphadenectomy for locally advanced gastric cancer. We performed the present study to compare the long-term outcomes of patients after laparoscopic or open gastrectomy plus D2 lymphadenectomy. METHODS: The clinicopathological data of 182 gastric cancer patients receiving gastrectomy plus D2 lymphadenectomy between January 2011 and December 2015 at Shenzhen Traditional Chinese Medicine Hospital were retrospectively retrieved. The overall survival (OS) and disease-free survival (DFS) of these 182 patients were compared. Then, the prognostic significance of positive lymph node ratio (LNR) was assessed. RESULTS: As a whole, OS (P = 0.789) and DFS (P = 0.672) of patients receiving laparoscopic gastrectomy plus D2 lymphadenectomy were not significantly different from those of patients receiving open surgery. For stage I patients, laparoscopic gastrectomy plus D2 lymphadenectomy was not significantly different from open surgery in terms of OS (P = 0.573) and DFS (P = 0.157). Similarly, for stage II patients, laparoscopic gastrectomy plus D2 lymphadenectomy was not significantly different from open surgery in terms of OS (P = 0.567) and DFS (P = 0.830). For stage III patients, laparoscopic gastrectomy plus D2 lymphadenectomy was not significantly different from open surgery in terms of OS (P = 0.773) and DFS (P = 0.404). Laparoscopic or open gastrectomy plus D2 lymphadenectomy was not proven by Cox regression analysis to be an independent prognostic factor for OS and DFS. High LNR was significantly associated with worse OS (P < 0.001) and DFS (P < 0.001). Surgical type did not significantly affect prognosis of patients with low LNR or survival of patients with high LNR. CONCLUSIONS: For patients with gastric cancer, laparoscopic gastrectomy plus D2 lymphadenectomy was not inferior to open surgery in terms of long-term outcomes. LNR is a useful prognostic marker for GC patients.

Schiff-Linked PEGylated Doxorubicin Prodrug Forming pH-Responsive Nanoparticles With High Drug Loading and Effective Anticancer Therapy.

Developing efficacious drug delivery systems for targeted cancer chemotherapy remains a major challenge. Here we demonstrated a kind of pH-responsive PEGylated doxorubicin (DOX) prodrug via the effective esterification and Schiff base reactions, which could self-assemble into the biodegradable micelles in aqueous solutions. Owing to low pH values inside the tumor cells, these PEG-Schiff-DOX nanoparticles exhibited high drug loading ability and pH-responsive drug release behavior within the tumor cells or tissues upon changes in physical and chemical environments, but they displayed good stability at physiological conditions for a long period. CCK-8 assay showed that these PEGylated DOX prodrugs had a similar cytotoxicity to the MCF-7 tumor cells as the free DOX drug. Moreover, this kind of nanoparticle could also encapsulate small DOX drugs with high drug loading, sufficient drug release and enhanced therapeutic effects toward MCF-7 cells, which will be benefited for developing more drug carriers with desirable functions for clinical anticancer therapy.